The objective of this project is to develop a practical state-of-the-art method for restriction endonuclease immobilization. The procedure is based upon use of a novel type of enzyme carrier: positively charged, colloidal 20-40 nm silica micro-beads. These beads are readily dispersible and can be used in reaction volumes of 20 Mul. The high density of the beads enables them to be rapidly sedimented in a microfuge. Eco RI will be used in this project, however once developed, the technique will be adapted to other important restrictions and ligation enzymes. The following approach will be evaluated: Enzyme and a non-catalytic protein such as BSA will be bound to the beads by electrostatic adsorption. The proteins will then be covalently coupled using a bifunctional cross-linking reagent such as glutaraldehyde. Coating all positively charged regions on the bead surface with BSA will prevent DNA from becoming adsorbed to beads. The use of an electrostatic adsorption technique for immobilization will ensure the complete binding of added enzyme. Since restriction enzymes have become so widely utilized in biomedical research, development of this system will have two advantages. First, it will enable the instantaneous separation of enzyme from substrate. This would be particularly beneficial when large numbers of digests are performed simultaneously. Secondly, it will become possible to reuse costly enzyme. In addition to these practical benefits, this study will provide increased understanding of the manner by which surface-bound enzymes interact with macromolecular substrates. This knowledge will be used to bio-engineer analogous systems, utilizing a diversity of biomedically important enzymes.